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Analysis of Determinants in Filovirus Glycoproteins Required for Tetherin Antagonism

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Analysis of Determinants in Filovirus Glycoproteins Required for Tetherin Antagonism Viruses 2014, 6, 1654-1671; doi:10.3390/v6041654 OPEN ACCESS viruses ISSN 1999-4915 www.mdpi.com/journal/viruses Article Analysis of Determinants in Filovirus Glycoproteins Required for Tetherin Antagonism † † † Kerstin Gnirß 1,2, , Marie Fiedler 1, , Annika Krämer-Kühl 1,2, ,‡, Sebastian Bolduan 3, Eva Mittler 4,#, Stephan Becker 4, Michael Schindler 3 and Stefan Pöhlmann 1,2,* 1 Infection Biology Unit, German Primate Center, 37077 Göttingen, Germany; E-Mails: [email protected] (K.G.); [email protected] (M.F.); [email protected] (A.K.-K.) 2 Institute of Virology, Hannover Medical School, 30625 Hannover, Germany 3 Institute of Virology, Helmholtz Center Munich, 85764 Neuherberg, Germany; E-Mails: [email protected] (S.B.); [email protected] (M.S.) 4 Institute of Virology, Philipps-University-Marburg, 35043 Marburg, Germany; E-Mails: [email protected] (E.M.); [email protected] (S.B.) † These authors contributed equally to this work. ‡ Present address: Boehringer Ingelheim Veterinary Research Center GmbH & Co. KG, 30559 Hannover, Germany. # Present address: Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA. * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +49-551-3851-150, Fax: +49-551-3851-184. Received: 25 November 2013; in revised form: 27 March 2014 / Accepted: 30 March 2014 / Published: 9 April 2014 Abstract: The host cell protein tetherin can restrict the release of enveloped viruses from infected cells. The HIV-1 protein Vpu counteracts tetherin by removing it from the site of viral budding, the plasma membrane, and this process depends on specific interactions between the transmembrane domains of Vpu and tetherin. In contrast, the glycoproteins (GPs) of two filoviruses, Ebola and Marburg virus, antagonize tetherin without reducing surface expression, and the domains in GP required for tetherin counteraction are unknown. Here, we show that filovirus GPs depend on the presence of their authentic Viruses 2014, 6 1655 transmembrane domains for virus-cell fusion and tetherin antagonism. However, conserved residues within the transmembrane domain were dispensable for membrane fusion and tetherin counteraction. Moreover, the insertion of the transmembrane domain into a heterologous viral GP, Lassa virus GPC, was not sufficient to confer tetherin antagonism to the recipient. Finally, mutation of conserved residues within the fusion peptide of Ebola virus GP inhibited virus-cell fusion but did not ablate tetherin counteraction, indicating that the fusion peptide and the ability of GP to drive host cell entry are not required for tetherin counteraction. These results suggest that the transmembrane domains of filoviral GPs contribute to tetherin antagonism but are not the sole determinants. Keywords: tetherin; ebola; lassa; glycoprotein 1. Introduction Research conducted in the last 15 years revealed that host cells can express antiviral effector molecules, which inhibit spread of certain viruses by interfering with discrete steps in the viral life cycle [1,2]. The expression of these virus-restricting factors, short restriction factors, can be constitutive but is frequently inducible by interferon (IFN). Single restriction factors have been identified by screening approaches seeking to determine the molecular mechanism underlying a specific antiviral activity [3–7]. A recent systematic screen for IFN-induced antiviral effectors provided detailed insights into the full restriction factor repertoire of the host cell [8]. One of the restriction factors uncovered in this screen [8] and previously identified as a cellular protein which inhibits release of HIV-1 [5] was the transmembrane protein tetherin (BST-2, CD317). It is now appreciated that tetherin restricts the release of several enveloped viruses and recent findings suggest a significant contribution of tetherin to retrovirus control in experimentally infected mice [9,10]. Tetherin’s unusual membrane topology is the basis for its antiviral activity. Tetherin contains two membrane anchors, a transmembrane domain at the N-terminus and a C-terminal GPI anchor [11]. It has been proposed that the latter might meet the criteria of a transmembrane domain more than those of a GPI anchor [12]. As a consequence of the two membrane-anchoring domains, tetherin can simultaneously insert into the plasma membrane and the viral membrane, thereby forming a physical connector between host cell and virus, which prevents release of progeny particles into the extracellular space [13,14]. The importance of tetherin’s domain organization for its antiviral activity is highlighted by two observations: Mutation of either membrane-anchoring domain interferes with inhibition of viral release and artificial tetherin molecules, which bear no sequence similarity with wt tetherin but exhibit the same domain organization, display antiviral activity [13]. In order to ensure efficient spread in tetherin positive cells, several viruses encode proteins which antagonize the antiviral activity of tetherin, mainly by removing tetherin from the site of viral budding. The first tetherin antagonist identified was the HIV-1 accessory protein Vpu [5]. Vpu antagonizes tetherin by removing it from the plasma membrane [15]. This process depends on interactions between the transmembrane domains of Vpu and tetherin [16–19], which allow for a Vpu-dependent alteration of tetherin trafficking. Thus, Vpu interactions with tetherin prevent transport of newly synthesized or Viruses 2014, 6 1656 recycled tetherin to the plasma membrane [16,20]. In addition, Vpu can remove tetherin from the plasma membrane [16,20,21], although with relatively low efficiency, and can induce tetherin ubiquitination and degradation in endolysosomes [22–24]. However, the contribution of these processes to tetherin antagonism by Vpu seems to be minor. Finally, it is notable that species specific differences in the amino acid sequence of the tetherin transmembrane domain can alter susceptibility to antagonism by Vpu [11], underlining that the tetherin interaction with Vpu is highly specific. The glycoproteins (GP) of ebolaviruses and marburgviruses, members of the Filoviridae family and highly pathogenic to humans, also antagonize tetherin, allowing efficient release of retro- and filovirus-like particles from tetherin transfected cells [25,26]. In addition, the tetherin antagonism by GP is likely responsible for the very modest [26] or absent [27] inhibition of release of authentic Ebola virus (EBOV) by tetherin. Notable differences between tetherin antagonism by Vpu and EBOV-GP have been reported. A direct interaction of the GP2 subunit of EBOV-GP and tetherin has been documented [26] but might be dispensable for tetherin antagonism, since EBOV-GP unlike Vpu can counteract artificial tetherin [28]. Similarly, EBOV-GP but not Vpu antagonizes tetherin orthologues from different mammalian species [26]. Moreover, EBOV-GP in contrast to Vpu does not reduce tetherin expression at the cell surface [26,28] and does not remove tetherin from lipid rafts [28,29]. In sum, EBOV-GP and Vpu employ markedly different mechanisms to counteract tetherin and the determinants in GP, which govern tetherin counteraction, are largely unclear. Lassa virus (LASV), a member of the Arenaviridae family and like EBOV highly pathogenic to humans, is inhibited by tetherin and the viral glycoprotein (GPC) does not antagonize tetherin [27,30]. A previous study constructed chimeras between the Marburg virus GP (MARV-GP) and LASV-GPC in order to identify determinants of viral assembly [31]. Here, we employed these LASV-GPC/MARV- GP chimeras and newly constructed LASV-GPC/EBOV-GP chimeras to map domains important for tetherin counteraction. We found that the cytoplasmic tail of EBOV- and MARV-GP and the integrity of the fusion peptide sequence in EBOV-GP are dispensable for tetherin counteraction. The exchange of the transmembrane or cytoplasmic domains between EBOV-GP/MARV-GP and LASV-GPC impeded tetherin counteraction by the filovirus GPs and did not transfer tetherin antagonism to LASV-GPC, indicating that the determinants in GP, which govern tetherin antagonism, are complex. 2. Results 2.1. LASV-GPC Fails to Antagonize Tetherin in the Context of a HIV-1 Gag-Based Virus-Like Particle System We sought to employ chimeric EBOV/MARV and LASV glycoproteins to identify domains important for tetherin counteraction. As a prerequisite to these studies, we first determined tetherin counteraction by the wt proteins. Expression of EBOV-GP, MARV-GP and LASV-GPC with a C-terminal V5 antigenic tag was readily detectable by Western blot analysis of transfected 293T cells (Figure 1A). As expected, all precursor glycoproteins and their mature C-terminal transmembrane units, which are generated upon processing of the precursors by host cell proteases, were detected and largely exhibited the expected molecular weights [32–37]. Moreover, a lentiviral vector pseudotyped with these glycoproteins (without V5 tag) was able to efficiently transduce 293T cells while no Viruses 2014, 6 1657 appreciable transduction was observed upon inoculation of cells with a vector bearing no glycoprotein (Figure 1B). Thus, the constructs available for our study allowed for robust glycoprotein expression and the glycoproteins were able to mediate efficient host cell entry, as expected. In order to determine whether the glycoproteins
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